Effector but not naïve regulatory T cells (Treg cells) can accumulate in the peripheral blood as well as the tumor microenvironment, expand during tumor progression and be one of the main suppressors for anti-tumor immunity. However, the underlying mechanisms for effector Treg cell expansion in tumor are still unknown. We demonstrate that effector Treg cell-mediated suppression of anti-tumor CD8+ T cells is tumor non-specific. Furthermore, TNFR2 expression is increased in these Treg cells by Affymetrix chip analysis which was confirmed by monoclonal antibody staining in both hepatocellular carcinoma and colorectal cancer patients and murine models. Correspondingly, increased levels of TNF-α in both tissue and serum were also demonstrated. Interestingly, TNF-α could not only expand effector Treg cells through TNFR2 signaling, but also enhanced their suppressive activity against anti-tumor immunity of CD8+ T cells. Furthermore, targeting TNFR2 signaling with a TNF-α inhibitor could selectively reduce rapid resurgence of effector Treg cells after cyclophosphamide-induced lymphodepletion and markedly inhibit the growth of established tumors. Herein, we propose a novel mechanism in which TNF-α could promote tumor-associated effector Treg cell expansion and suggest a new cancer immunotherapy strategy using TNF-α inhibitors to reduce effector Treg cells expansion after cyclophosphamide-induced lymphodepletion.